Photodynamic cancer treatment based on bioluminescence resonance energy transfer

Abstract

The use of photodynamic therapy (PDT) at malignant and premalignant lesions has been on the increase. However, PDT is ineffective to treat metastatic and large size tumors due to delivery limitation of light itself. To overcome these problems, we introduced the PDT using bioluminescence resonance energy transfer (BRET) because bioluminescence can activate the photosensitizers at disease lesions that light cannot penetrate owing to enzyme-substrate reaction. Emission wavelength of coelenterazine which is substrate of RLuc8 does not matched with excitation wavelength of second generation photosensitizer such as chlorin e6. So, 655nm quantum dots were conjugated with RLuc8 using BRET. After coelen-terazine administration, quantum dots conjugated with RLuc8 emit the specific overlapped wavelength light with absorption wavelength of chlorin e6. BRET PDT generated similar amounts of reactive oxygen species compared to conventional PDT and induced significant cytotoxicity in vitro experiments. And degree of cytotoxicity in BRET PDT was similar that of conventional PDT. In spite of tiny light of bioluminescence, significant cytotoxicity in BRET PDT is supposed to energy transfer between photosensitizer inside cell membrane and quantum dots conjugated with RLuc8 attached at cell membrane. In addition, reactive oxygen species generated during BRET PDT at cell membrane induce lipid peroxidation. In vivo experiments, BRET PDT group had tendency to inhibit growth of tumor compared to control group. In addition, there were significant apoptotic lesions and decreased tumor vessels inside the tumors in immunofluorescence assay. Furthermore, popliteal lymph node where conventional cannot delivered were treated using BRET PDT at footpad tumor injec-tion model. As treating popliteal LN which is moving route of tumors, lung metastasis was decreased after BRET PDT compared to control. In conclusion, BRET PDT may be novel method to overcome the limitations of conventional PDT and to prevent distant metastasis. BRET phenomenon will be applied other applications such as optogenetics.